1. Field of the Invention
This invention pertains generally to fluidic devices, and more particularly, to fluidic nanotubes and devices fabricated therefrom.
2. Description of Related Art
Sensors utilizing novel nanostructured materials and new mechanisms offer to significant impact a broad range of applications relating to national security, health care, the environment, energy, food safety, and manufacturing. Emerging micro- and nano-technologies can decrease the size, weight and cost of sensors and sensor arrays by orders of magnitude, and increase their spatial and temporal resolution and accuracy.
There are urgent civilian and military needs at this moment for new sensors and sensor systems which include: (1) the ability to respond to new toxic chemicals, explosives and biological agents, (2) providing enhanced sensitivity, selectivity, speed, robustness, and immunity from false alarms, and (3) the ability to function, perhaps autonomously, in unusually complex environments (NSF 03-512). In this regard, the design and synthesis of functionalized nanostructured materials and development of new sensing mechanisms could play a significant role in the process of developing efficient chemical and biological sensors.
In general, ideal nanostructured materials would have some degree of porosity/high surface area with suitable analyte interaction mechanism employing various sensing principles such as mechanical, chemical, electrical, chromatographic, biological, fluidic, optical, and mass sensing.
Since the discovery of carbon nanotubes in 1991, there have been significant research efforts devoted to nanoscale tubular forms of various solids. The formation of a tubular nanostructure generally requires a layered or anisotropic crystal structure. There are reports of nanotube formation of solids lacking layered crystal structures such as silica, alumina, silicon and metals through templating of carbon nanotubes and porous membranes or thin film rolling. Such nanotubes, however, are either amorphous, polycrystalline or exist only in ultra high vacuum.
Hollow inorganic nanotubes are attracting a great deal of attention due to their fundamental significance and potential applications in bioanalysis and catalysis. Among them, silica nanotubes are of special interest because of their hydrophilic nature, easy colloidal suspension formation, and surface functionalization accessibility for both inner and outer walls. Such modified silica nanotubes and nanotube membrane have shown potential applications for bioseparation and biocatalysis. Recently, bright visible photoluminescence from sol-gel template synthesized silica nanotubes was observed. In addition, the study of the physical and chemical nature of molecules or ions confined within the inorganic nanotubes is of great current interest.
Silica nanotubes have been synthesized typically within the pores of porous alumina membrane templates using a sol-gel coating technique. Alumina templates can be dissolved to liberate single silica nanotubes. Such nanotubes, which are prepared at low temperature, have porous walls and are relatively fragile. Once the templates are removed, the silica nanotubes will generally bundle up and become less oriented. The same applies to the silica nanotubes prepared at low-temperature using other templates.
Over the years, various molecular detection techniques have been developed for their chemical/biological sensing, diagnostic and prognostic utility. For most, efficiency is a result of a trade-off between sensitivity, specificity, ease of operation, cost, speed and immunity to false alarms. Novel functional materials such as quantum dots, photonic crystals, nanowires, carbon nanotubes, porous membranes, porous silicon and sol-gel matrices incorporating biomolecules have been used as sensing elements with various possible detection mechanisms. For example, the use of quantum dots has been demonstrated as fluorescent biological labels with several advantages over traditional organic dyes. Major advantages of this approach are the high extinction coefficient, bright wavelength-tunable fluorescence and superior photostability of the quantum dots. Metal nanoparticles have also been utilized for various biological sensing applications with significant enhanced sensitivity and specificity. The ability of porous silicon to display well-resolved Fabry-Perot fringes for biosensing applications has been utilized in this regard, and molecular imprinted sol-gel process for biosensing has been recently developed. In addition, a new sensing scheme has been developed using swellable photonic crystals as active components.
In addition to these efforts, one-dimensional nanostructures (nanotubes and nanowires) have recently received significant attention as possible miniaturized chemical and biological sensing elements. The ultrahigh surface to volume ratios of these structures make their electrical properties extremely sensitive to surface-adsorbed species, as recent work has shown with carbon nanotubes, functionalized silicon nanowires and metal nanowires.
Chemical and biological nanosensors are interesting because of their potential for detecting very low concentrations of biomolecules or pollutants on platforms small enough to be used in vivo or on a microchip. For example, a room temperature photochemical NO2 sensor has recently been demonstrated based on individual single-crystalline oxide nanowires and nanoribbons. Regardless of their nanotube or nanowire morphologies, the sensing mechanism generally used in these studies is the electron-transfer process between the analytes (in solution or gas) and the nanostructures, thus inducing conductivity changes. It has been demonstrated in the field that for metal nanowire sensing, different mechanisms have to be invoked on the metal nanowires. These one dimensional nanostructures generally provide excellent sensitivity due to having an inherent high surface to volume ratio. However, the sensing selectivity for these structures has been less than ideal, although in many cases this can be improved by surface functionalization of specific receptors.
Chemical/sensing systems are being studied using silica and gold tubular membranes. These membranes represent a new class of molecular sieves for molecular separation and electrochemical sensing based on the size of the molecules as well as interaction with the tubes surface functional group. In most of these studies, the inorganic nanotube membrane (polycarbonate or porous alumina) was set up to separate two salt solutions and a constant transmembrane potential was applied, then the transmembrane current was measured. When an analyte of comparable dimensions to the tube diameter was added to one of the solutions, a decrease in transmembrane current was sensed because of the current blocking by the molecules. Using such schemes, ultratrace of different ions and molecules were detected. These experiments, however, have all relied on using entire membranes as sensing elements. No significant efforts have been placed on single tube sensing, although the use of single nanotube sensing would obviously represent the miniaturization limit.
It is also worth noting that recently developed artificial nanopores have been fabricated using soft lithography or ion mill to carry out molecular sensing through individual nanotubes. These processes are subject to the problem of scaling up or the pore size limitation (i.e. 200 nm for the PDMS approach). The use of carbon nanotubes for this type of nanofluidic sensing applications has also been proposed. A number of significant technical hurdles, however, need to be overcome before these can become a viable nanofluidic sensing element. Examples of these hurdles include: the difficulty of surface functionality (both internally and externally), and the difficulty associated with control over the metallic tube versus semiconductor tubes.
Capillary Electrophoresis (CE) is a technique similar to gel electrophoresis with an added advantage of smaller sample consumption (<10 nL), automation, faster analysis and integration with an on-line detection system. The high surface to volume ratio of the capillary allows the application of high voltage to achieve fast separation with efficient heat dissipation to prevent band-broadening effects. The ends of the capillary are in contact with reservoirs filled with the electrolyte, where electrical potential can be applied through non-reactive electrodes. UV absorbance, laser induced fluorescence and electrochemical detections (e.g. potentiometric) can be used on-line for detection of separated molecules in CE. The application of CE to detect sialic acids in serum as a tumor marker has been demonstrated. Even though the cost per run for CE is low, the initial cost of instrumentation and detection systems can be prohibitive.
FIG. 1 and FIG. 2 illustrate a 120 mV bias across an α HL ion channel which produces an ionic current of ≈120 pA. When a single polynucleotide strand passes through the channel, the current drops to 15-50 pA. The amplitude of the current drop and its duration depend on the type of nucleotide.
Previous work on nanopore based single molecule detection can be broadly classified into two categories, namely: (i) non-functionalized nanopores; (ii) functionalized nanopores. Almost all of the prior work has involved the transmembrane protein ion channel α-Hemolysin (α HL) embedded in a suspended membrane separating two chambers filled with ionic solution. The entrance on the top (cis) side is about 2.6 nm in diameter whereas the narrow channel through the membrane that is closer to the bottom end (trans) is 1.4 nm in diameter. When a voltage bias of 120 mV is applied across the ion channel, an ionic current of about 120 pA is produced for ionic concentrations of 1 MKCl (the resistance is approximately 109 Ω).
When single-stranded polynucleotides are introduced in one of the chambers, they electrophoretically flow through the ion channel. By doing so, they block the ionic current, which reduces to levels of 15-50 pA as seen in FIG. 2. The time of flight of these polynucleotides seems to vary linearly with their length, and inversely with the applied voltage. It has been hypothesized that different nucleotides would have different blocking signatures (either time of flight or amplitude of current drop), which would allow one to rapidly sequence ssDNA directly. This has led to many attempts over the last decade, and there has been partial success in discriminating between different bases. For example, polycs seem to produce shorter but deeper (lower current) decrease in ionic current whereas polyAs produce longer but shallower reductions. However, direct and rapid sequencing of ssDNA has been unsuccessful and remains a challenge, although hairpin DNA molecules have been detected with single nucleotide resolution.
One of the problems in direct sequencing arises from the fact that the time a single base spends in the nanopore is too short and that the number of ions that it blocks is too few (i.e. approximately 100), making it difficult to detect it above the background noise. Slowing down the polynucleotides could offer a chance of direct sequencing, but that has also remained a challenge. More recently, artificial nanopores have been demonstrated that can be fabricated from inorganic materials, and that show similar behavior in blocking ionic current when ssDNA passes through them. However, direct sequencing of ssDNA has not been reported so far and parallel processing of those artificial nanopores has proven to be very difficult with this approach.
While it has so far been very difficult to achieve biomolecule specificity using non-functionalized nanopores, recent work of using functionalized α HL nanopores has shown promise. Nanopores have been functionalized using a ssDNA probe attached at the cis entrance through a disulphide linkage to a cysteine residue in the α HL protein.
FIG. 3A and FIG. 3B illustrate a probe ssDNA which is attached through a disulphide linkage to a cysteine residue at the cis opening of a α HL nanopore protein as schematically shown in the upper portions of FIG. 3A and FIG. 3B. The lower halves of FIG. 3A and FIG. 3B depict time traces of the ionic current passing through the nanopore. If the complementary target ssDNA is transported through the nanopore as shown in FIG. 3A, it binds with the probe strand which reduces the ionic current for approximately 50 mS. However, if a single base pair mismatch is introduced as shown in FIG. 3B, the binding lifetime is reduced to about 1 mS.
Then by transporting target ssDNA sequences, they found that when the target was fully complementary, its residence time in the nanopore, as measured by the duration of the reduced ionic current, was much longer (≈50 mS) than if even a single base-pair mismatch is introduced (≈1 mS). From this, kinetics of the binding reaction can be quantified. Furthermore, the α HL protein nanopore has been functionalized with other molecules to study reaction kinetics of various molecular interactions such as small molecules with proteins, ions with proteins, and so forth. The use of functionalized nanopores for biomolecular analysis could yield a number of benefits, however, such efforts have met with fabrication difficulties.
Therefore, a need exists for nanofluidic devices and nanotube structures which can be readily implemented, such as within fluidic sensing applications. The present invention fulfills those needs and others, while overcoming the drawbacks inherent in prior nanodevice and nanostructure approaches.